Granulysin is a 9 kD a-helical protein expressed by human natural killer cells and T lymphocytes. Recombinant granulysin lyses both mammalian cells and a broad spectrum of microbes. Synthetic peptides (10-30 residues) corresponding to the central region of granulysin recapitulate its lytic activity. In a subset of these peptides, replacement of cysteine or arginine residues, or introduction of D-amino acids to disrupt the a-helix, results in the loss of activity against mammalian cells with little or not effect on antimicrobial activity. The goal of this Project is to evaluate candidate immunotherapeutics based on the granulysin sequence for biodefense and for treatment of antibiotic resistant organisms. The specific aims are: 1) assay in vitro all first generation substituted peptides generated in Project 1 for activity against Vibrio cholerae, Salmonella typhimurium, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, Staphlococcus aureus, Mycobacterium tuberculosis, human lymphocytes, and human erythrocytes; 2) assay in vitro all second generation derivative compounds produced in Project 1 on the same panel of microbes as well as on a panel of antibiotic resistant clinical isolates; and 3) evaluate the most promising compounds in vivo in three models: topical application in a murine wound model of infection; intragastric or intraintestinal application in the infant mouse and rabbit ileal loop models of cholera; and aerosol treatment of inhaled Mtb in a mouse model. The dose, kinetics, and synergy with conventional antibiotics will be assessed. Results from this Project should identify promising candidate compounds that can be advanced to clinical trials.